It has been demonstrated earlier that electrophilic compounds present in vivo can be monitored by measuring the products (adducts) of their reaction with proteins, in particular hemoglobin (Hb) (1-5). Important nucleophilic sites in proteins, e.g., hemoglobin (Hb), which are reactive under physiological conditions are the imidazole nitrogen atoms in histidine residues, sulfur atoms in cysteine and methionine residues, oxygen atoms in carboxyl groups and in hydroxyl groups in tyrosine and serine residues, and the α-nitrogen atoms in the N-terminal valine residue of all four chains of human Hb (6).
The so-called N-alkyl Edman procedure was developed for measurements of adducts (mainly low-molecular weight adducts) to N-terminal valine residues in Hb (7). This method was based on the original Edman degradation procedure (8,9) used for protein sequencing. It was observed that N-terminal valine N-alkylated with a radioactively labeled 2-hydroxyethyl moiety from ethylene oxide was released spontaneously as a phenylthiohydantoin (PTH) under the conditions (pH>7) employed for the coupling reaction between phenyl isothiocyanate (PITC) and protein. The released PTH could be separated from unmodified N-terminal valine residues, as well as from the rest of the protein by means of extraction.
This observation led to the development of the N-alkyl Edman procedure for gas chromatographic (GC)/mass spectrometric (MS) determination of Hb adducts (10). Because of its usefulness, the N-alkyl Edman method has been applied in a number of laboratories for research purposes, dose monitoring and hygienic surveillance (11-16).
A brief description of the N-alkyl Edman procedure is presented in FIG. 1. A sample of the globin (isolated from red blood cells by acid precipitation) is dissolved in formamide. Pentafluorophenyl isothiocyanate (PFPITC) is then added, together with a small amount of aqueous 1 M NaOH in order to obtain a near-neutral solution. This mixture is maintained at room temperature overnight, after which the temperature is raised to 45° C. for approximately two hours (17). The pentafluorophenylthiohydantoin (PFPTH) derivatives of the terminal N-alkylvaline residues are released in high yield by this procedure and can subsequently be isolated by extraction (liquid-liquid).
Although the N-alkyl Edman procedure has become an established method for analysis of N-substituted hemoglobin adducts, the method has its restrictions, e.g., the range of adducts that can be analyzed is limited. Small adducts, e.g., involving ethylene oxide and propylene oxide, can be quantified at the pmol/g globin level, which is sensitive enough for measurement of background adduct levels (levels without known exposure). However, adducts with a few polar groups are more difficult to analyse, due to the limitations imposed by the GC separation system prior to MS detection. Some of these limitations can be solved, e.g., by further derivatisation of the polar groups (18). However, this approach is both time-consuming and demands the development of new procedures for each specific adduct. Furthermore, adducts of high molecular weight (>500 mass units, mu) and/or thermolabile adducts are extremely difficult to analyze using the GC-MS based N-alkyl Edman procedure.
Accordingly, there is a need for more versatile and sensitive, as well as simpler methods for analyzing adducts.